Understanding the complex mechanisms that control bone formation and bone turnover has major medical implications. Osterix (Osx), which was discovered in our laboratory, is an essential transcription factor in osteoblast differentiation. During bone formation in the embryo Osx commits precursor cells to become fully functioning osteoblasts, which synthesize and secrete the components of the bone matrix. Based on our preliminary results, we hypothesize that Osx continues to have a critical role in osteoblast and osteocyte function beyond embryonic development in the physiological control of bone homeostasis postnatally. To understand this role of Osx after birth, mice have been generated in which Osx is inactivated postnatally. Bones of these mice will be extensively characterized by microCT, histomorphometry and electron microscopy methods as well as by quantitation of molecular markers of osteoblasts, osteocytes and osteoclasts. The mutant mice will also test the hypothesis that Ox is a negative regulator of canonical Wnt signaling in osteoblasts. We have recently identified the chromatin protein NO66 as the first negative regulator of the transcriptional activity of Osx. NO66, which binds to Osx, has a demethylase activity that is targeted to the methylated forms of histone H3K4 and H3K36 markers of transcriptionally active chromatin. This specific histone demethylase activity is needed for the inhibition of the transcription activation function of Osx by NO66. Based on knockdown experiments in osteoblasts we hypothesize that the chromatin protein NO66 has a critical physiological function in osteoblasts. Characterization of mouse mutants in which NO66 is either conditionally inactivated or conditionally overexpressed in Osx expressing cells, is essential to understand the physiological role of NO66 in bones. To gain a mechanistic understanding of the relationship between Osx and NO66, we also propose to examine the dynamics of Osx and NO66 occupancy in the chromatin of Osx target genes during osteoblast differentiation and to investigate whether the dynamics of Osx occupancy in the chromatin of Osx target genes are disrupted in osteoblasts of mice in which NO66 is either conditionally inactivated or overexpressed in these cells. Overall, the proposed experiments should provide considerable new insights in the mechanisms of bone formation by Osx in the homeostatic control of bone remodeling. PUBLIC HEALTH RELEVANCE: Understanding the mechanisms by which bone formation is controlled, is of critical importance for identifying targets for appropriate therapies for bone diseases. Osterix is a transcription factor that is completely required for bone formation during embryonic development. Our hypothesis is that Osterix is also required for the synthesis of components of the bone matrix after birth and throughout life and for the normal function of osteocytes, the cells that are embedded in the bone matrix. We also hypothesize, that in addition Osterix controls the activity of specific signals of cell proliferation in bones. In another part of this project we will test the hypothesis that a specific chromatin protein, which we recently identified, controls the activity of Osterix in bones.